The underlying premise of this project is that through the use of chemical maneuvers, such as the introduction of specific cross-links or the proteolytic cleavage of intact lipoproteins, unique and detailed information can be obtained regarding protein structure in these particles. In order to obtain defined lipoprotein species for these studies, preparative isoelectric focusing will be employed to isolate major subfractions of human high density lipoproteins. The quaternary structure of these subfractions will then be studied by cross-linking with imidoester reagents. The tendency for mixtures of apolipoproteins to form hybrids with quaternary structures similar to the intact lipoproteins will also be studied by this method. The tertiary structure of the A-1 protein will be investigated in both its native and delipidated states by analysis of points of cross-link attachment; this information will assist in understanding the conformational changes which occur on lipid binding. Special efforts will be made to define areas of amino acid sequence in the apolipoproteins which are responsible for interaction with lipid. In one approach, photoactivatable cross-linking agents will be used to cross-link radioactive aminophospholipids to apoprotein side chains in intact HDL3. Specific sites of attachment will be determined by enzymatic proteolysis of isolated apoproteins and identification of peptides containing covalently attached aminophospholipids. In another study, enzymatic proteolysis of intact HDL3 will be used to generate a residual lipoprotein particle; determination of the peptides remaining associated wth lipid in this particle should provide insight into structural features required for lipid binding. Taken ensemble, these data will be used to evaluate various proposed models of lipoprotein structure.